Study Design

1. Multiple Sequence Alignment Sequence Identification Laboratory Methods Isolate DNA Edit Alignment Screen 16S amplicon Ligate Vector Tree Search in RAxML PCR Transform & Plate High Throughput Sequencing Molecular Microbiological Analysis of Severe Caries in Primary Teeth E.L. Gross*, E.J. Leys, B. Vriesendorp, N.D. Firestone, D.A. Janies, J.S. Martin, B.J. Paster, and A.L. Griffen Dental caries is the most common chronic disease of childhood, but we do not have a complete understanding of its complex microbial etiology. Dental plaque contains several hundred different organisms and many are poorly studied. In addition, comparatively little attention has been paid to identifying health-associated bacteria. Objectives: Our purpose was to identify bacterial species present in health and established severe caries of the primary dentition using an open-ended molecular approach. Methods: Plaque samples were collected from 32 subjects with severe caries, age 2 to 6, and 30 healthy controls matched by age, race, and sex. A plaque sample was taken from each of four sites in each caries subject: intact enamel, whitespots, the surface of cavitated lesions, and dentin. Quantitative clonal analysis of bacterial 16S ribosomal genes isolated from the plaque samples was used to identify bactieral taza. Phylogenetic trees based on likelihood models using bootstrap cutoffs were used to define taxa. Clinical data were analyzed by repeated measures ANOVA. Results: Resulting taxonomic designations were not always in agreement with traditional species designations. Streptococcus mutans and several other streptococci were found at high levels and increased as caries progressed. Multiple Lactobacillus species were associated with more advanced caries, and several were found at high levels Propionibacterium australiense was also associated with advanced caries. Health-associated species found at high levels included Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis, as well as Stretococcus gordonii, Streptococcus cristatus, and the Streptococcus intermedius/anginosus/constellatus group. Multiple additional taxa found at lower levels were more common in health than in caries. Conclusions: Many Streptococcus and Lactobacillus species are consistently found at high levels in subjects with established severe caries of the primary dentitition. There are many health-related species that appear to be lost as caries progresses. Supported by NIH DE16125 and F30DE019339. Abstract Clinical Methods Bioinformatics Methods Results • Study Design • Plaque samples have been acquired from 32 children with severe caries and 30 healthy controls • Inclusion Criteria for Caries Group • Presence of 4 primary teeth with smooth surface lesions, at least 2 of which involve the pulp • Inclusion Criteria for Healthy Control Group • Free of caries • No existing restorations • Matched to the caries group on age, race, and gender • Plaque samples were taken from • intact enamel • whitespot lesion • cavitated lesion • deep dentin Figure 1 Estimates by PROC MIXED of the percentage of change as caries progresses. * p<0.05. ** p<0.001. Figure 2 (A) Mean values for taxa that increase with caries progression (B) Mean values for taxa that decrease with caries progression Progression of Caries: Four Sites Dental caries is the most common chronic disease of childhood, but we do not have a complete understanding of its complex microbial etiology. Investigation of the pathogens associated with the onset and progression of severe caries could provide targets for biological interventions. Identification of beneficial health-associated species could provide the basis for therapeutic interventions to establish caries-resistant microbial communities. Introduction Enamel Whitespot Cavitation Deep Dentin Conclusions Local Curated Oral Microbiome Database of Supported OTU’s • S-OTU’s that increase as caries progresses: • S. mutans, Lactobacillus species, S. vestibularis-salivarius, P. australiense, Selenomonas sp oral clone CI-CS003, P. denticolens, M. micronuciformis, A. parvulum, and N. bacilliformis • There are many health-associated S-OTU’s that are lost as caries progresses To identify the oral bacteria associated with dental caries and health in the primary dentition using an open-ended molecular approach. Purpose